Glial cells in the mammalian central nervous system (CNS) that express the NG2 proteoglycan (NG2 cells) appear during late embryonic stages and rapidly expand to uniformly occupy the entire CNS. These cells are distinct from mature oligodendrocytes, astrocytes, or resting ramified microglia and represent a fourth major glial population. Cultured NG2 cells give rise to oligodendrocytes and are thus called oligodendrocyte precursor cells (OPCs). In vivo fate mapping studies using our newly generated NG2creBAC transgenic mice revealed that NG2 cells in the white matter and dorsal forebrain generate exclusively oligodendrocytes, while those in the gray matter of the ventral forebrain generate both oligodendrocytes and protoplasmic astrocytes. When the basic helix-loop-helix transcription factor Olig2 is deleted in NG2 cells, almost all the NG2 cells in the dorsal forebrain differentiate into astrocytes at the expense of oligodendrocytes, resulting in myelin loss. Thus, NG2 cells retain lineage plasticity, and a single transcription factor Olig2 plays a critical role in maintaining the oligodendroglial fate of NG2 cells. Deletion of Olig2 in more differentiated oligodendrocytes does not alter the level of neocortical GFAP expression, in contrast to Olig2 deletion at earlier stages, suggesting that there is a developmental window in which oligodendrocyte lineage cells can be converted into astrocytes in the absence of Olig2. Aim 1 will test the hypothesis that NG2 cells lose their lineage plasticity and become incapable of generating astrocytes as they mature into oligodendrocytes, and that restriction of lineage plasticity in NG2 cells is regulated by epigenetic mechanisms. Aim 2 will test the hypothesis that there is a signaling pathway that maintains the expression of Olig2 in NG2 cells in the normal brain and prevents them from differentiating into astrocytes. This will be explored by screening small-molecule libraries for a compound that alters Olig2 transcriptional activity. Identified compounds will be used in future studies to delineate the endogenous signaling pathways that regulate Olig2 expression. The pathway could then be manipulated in future injury repair paradigms to promote differentiation of NG2 cells into the desired cell type. PUBLIC HEALTH RELEVANCE: NG2 cells represent a glial progenitor population that is ubiquitously distributed throughout the gray and white matter of the central nervous system. The proposed studies are aimed to identify the mechanisms that regulate their lineage plasticity through the transcription factor Olig2. The results from these studies can be used in future experiments in which the fate of endogenous NG2 cells can be manipulated to maximize their contribution to lesion repair in various types injury.